CAR is a junction adhesion molecule (JAM), similar to other known JAM proteins. These proteins generally localize to tight junctions, where they participate in cell-cell adhesion and link to cytoplasmic proteins. Several JAM proteins bind counter-receptors on leukocytes and participate in their transmigration across cell layers. JAM interactions with most leukocyte types have been reported, except for B cells. New data show that CAR binds B cells, thus establishing that JAM-leukocyte interactions include all major classes of white blood cells. New data also show that CAR binds immunoglobulins. Details of these new CAR activities remain to be elucidated. The long-term objective of this research is to identify the molecular interactions in which CAR participates and reach a more complete understanding of CAR function. In the sort-term, this project will identify the counter-receptor on B cells, cellular responses mediated by the CAR-B cell interaction, and the relationship of CAR-immunoglobulin binding to CAR function. Specific Aim 1. Identify the CAR counter-receptor present on B cells. Obtain cDNA for expression and isolation of recombinant protein. The B cell counter-receptor will be purified from Raji cells and identified by comparison of amino-terminal sequence with protein and genetic databases, cDNA encoding the protein will be obtained from a repository (if the protein is known and the cDNA available) or by cloning de novo. Specific Aim 2. Characterize the response of B cells following CAR binding. In isolated peripheral blood mononuclear cells, B cells express early markers of activation following binding by CAR. Isolated B cells will be tested for responsiveness to CAR, and components essential for the response will identified. In addition, CAR may enhance B cell transmigration through endothelial cell layers. CAR's role in leukocyte migration will be studied using cells that express wild type and recombinant forms of CAR in a Transwell migration system. Specific Aim 3. Characterize the CAR-immunoglobulin interaction. Specific domains of CAR and immunoglobulins that mediate their interaction will be identified and mapped to determine their structural relationships to regions of these proteins known to participate in other interactions. Interactions between CAR binding immunoglobulins and the B cell counter receptor will identified. This work will advance knowledge of cells that form tight _junctions and their interactions with cells and molecules that must cross those barriers.